1. Field of the Invention
The present invention relates to a method for purifying darbepoetin alfa by selectively separating only a structural isoform having a high content of sialic acid from a mixture of structural isoforms of darbepoetin alfa having various contents of sialic acid.
2. Description of the Related Art
Darbepoetin alfa (NESP) is an analogue of erythropoietin (EPO), which has five substitutes in the amino acid sequence of the erythropoietin molecule, providing two additional N-glycosylated chains (International Publication No. WO 2001076640). Although darbepoetin alfa differs from EPO in terms of biochemical characteristics such as molecular weight, isoelectric point, etc., it is also an erythropoiesis-stimulating protein like EPO.
Darbepoetin alfa is known to have a serum half-life which is about three times longer than that of erythropoietin in mice, rats, dogs, humans, etc., due to its high sialic acid content (Pedrazzoli P, Cinieri S, Lorusso V, Gamucci T, Secondino S, Silvestris N 2007 November-December, 27(6C), 4419-24; Anticancer Res.), and thus its in vivo decomposition is inhibited, thereby affording a higher biological activity than that of EPO in its natural state.
Darbepoetin alfa, due to the difference in sialic acid content by glycosylation, maximally has 22 different types of structural isoforms, and the higher the sialic acid content is, the lower the isoelectric point and the higher therapeutic value are. Accordingly, the selective separation and purification of only the structural isoforms with a high sialic acid content is very important in the therapeutic field using darbepoetin alfa.
Examples of the conventional method for purifying EPO and EPO analogues include anion exchange and cation exchange chromatographies, hydrophobic-interaction chromatography, size-exclusion chromatography, etc. Specifically, International Publication No. WO 2010/027869 discloses a method for purifying EPO by sequentially applying hydrophobic-interaction, anion exchange, cation exchange, and size-exclusion chromatographies. International Publication No. WO 2003/045996 discloses a method for purifying recombinant human EPO by performing a reverse phase chromatography, anion exchange chromatography, and size-exclusion chromatography.
In particular, as methods for purifying darbepoetin alfa, International Publication No. WO 1995/005465 discloses a method for applying anion exchange resin and C4 resin, and International Publication No. WO 2010/008823 suggests a flow-through mode for the purification of darbepoetin alfa having a high sialic acid content with an isoelectric point of 4.5 or less, in which the target protein is not bound to the column of the cation exchange resin but is flowed out in the chromatography treatment solution. However, these methods require extremely high cost and much time due to their use of various steps of resins, and are thus not suitable for large-scale production.
Meanwhile, Korean Patent Application Publication No. 10-2013-0042107 discloses a method which is more simplified than the above four-step process, by adopting a three-step process which sequentially applies anion, hydroxyapatite, anion exchange chromatography, and performing adsorption and washing under a particular pH condition when a secondary anion exchange resin chromatography is applied, for selective separation of isoforms having low isoelectric points. In particular, for obtaining isoforms with low isoelectric points, darbepoetin alfa is bound to the column at a pH ranging from 4.0 to 5.0 followed by washing with a buffer solution having a pH ranging from 2.0 to 2.4.
The above method may be advantageous in terms of time and cost required in large-scale production because the process is a bit simplified compared with the conventional processes, however, for the embodiment of the low pH condition (pH ranging from 2.2 to 2.4) as suggested in the above published patent application, it is necessary that a toxic, acidic solution such as HCl be used in a large amount, and thus this method is not desirable. Additionally, since the reactions of isoforms at a given pH condition are not constant when the amount to be treated by the column is increased at the time of scale-up, the possibility of reproducibility in large-scale production is low when isoforms with a desirable range of isoelectric points are to be obtained by controlling pH conditions of a buffer solution.
That is, since the conventional method of purifying darbepoetin alfa requires a complex process using chromatography consisting of various steps of resins, it requires an extremely high cost and much time, and when the purifying is performed using a process which is simplified compared with the conventional process, it is necessary that the conditions such as pH be precisely controlled to compensate for the purification effect that may be reduced, but it becomes more difficult to control the conditions as the production scale becomes larger.
As such, there is an increasing demand for an improved method of purifying darbepoetin alfa which, being more simplified than the conventional methods and having more easily controllable process conditions, can stably reproduce successful process conditions while reducing cost and time when the method is applied to large-scale production.